Sperm motility initiation, capacitation and hyperactivation are modulated by an interplay of intracellular Ca 2+, cyclic AMP and pH. Mechanisms by which these mediators alter sperm function have not been elucidated, but may involve reversible alterations in regulatory protein phosphorylation. Studies were designed to investigate the influence of the protein phosphatase (PP) inhibitor calyculin A (CL-A) on human sperm motility and to characterize the CL-A sensitive PP and its endogenous regulators in human and rhesus monkey sperm. CL-A (5-50 nM) treatment of human swim-up supernatants and pellets resulted in accelerated sperm velocity in both fractions and an increase in percent motility in pellet fractions. These results suggest that inhibition of PP1 or PP2A could be responsible for the motility stimulation, since these phosphatases are sensitive to nanomolar quantities of CL-A. PP activity in human (n=4) and rhesus monkey (n=4) sperm extracts were measured using [ 32P]phosphorylase-`, the preferred substrate for PP1 and PP2A, in the absence of divalent cations. Human (6.2 q 4.5 x10 -2 mU/10 6 sperm) and monkey (4.3 q 0.8 x 10 2 mU/10 6 sperm) extracts contained PP1 and/or PP2A activity. Sperm PP activity inhibition profiles by okadaic acid and CL-A were identical to those reported for PP1 derived from other tissue sources. Western blot analysis with antibodies against various isoforms of PP1 documented the presence of PP1 2 in human and monkey sperm. PP1 activity in most tissues is regulated by the heat-stable inhibitors I1 and I2. Sperm extracts contained inhibitor activity similar to I2, and glycogen synthase kinase-3 activity, which is involved in the activation of the inactive PP1-I2 complex. These results indicate, for the first time, that human and rhesus monkey sperm contain PP1, regulators of PP1 and that inhibition of PP1 activity by CL-A can enhance motility.